Garden bean named ‘210104’

ABSTRACT

A novel garden bean cultivar, designated ‘210104’, is disclosed. The invention relates to the seeds of garden bean cultivar ‘210104’, to the plants of garden bean line ‘210104’ and to methods for producing a bean plant by crossing the cultivar ‘210104’ with itself or another bean line. The invention further relates to methods for producing a bean plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other garden bean lines derived from the cultivar ‘210104’.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive Garden Bean(Phaseolus vulgaris) variety, designated ‘210104’. There are numeroussteps in the development of any novel, desirable plant germplasm. Plantbreeding begins with the analysis and definition of problems andweaknesses of the current germplasm, the establishment of program goals,and the definition of specific breeding objectives. The next step isselection of germplasm that possess the traits to meet the programgoals. The goal is to combine in a single variety or hybrid an improvedcombination of desirable traits from the parental germplasm. Theseimportant traits may include fresh pod yield, higher seed yield,resistance to diseases and insects, better stems and roots, tolerance todrought and heat, and better agronomic quality. With mechanicalharvesting of many crops, uniformity of plants characteristics such asgermination and stand establishment, growth rate, maturity and plantheight is important.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of plant breeding is to develop new, unique and superior gardenbean cultivars. The breeder initially selects and crosses two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. The breeder can theoretically generatebillions of different genetic combinations via crossing, selfing andmutations. The breeder has no direct control at the cellular level.Therefore, two breeders will never develop the same line, or even verysimilar lines, having the same bean traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsthat are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior garden bean cultivars.

The development of commercial garden bean cultivars requires thedevelopment of garden bean varieties, the crossing of these varieties,and the evaluation of the crosses. Pedigree breeding and recurrentselection breeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents that possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Garden bean, Phaseolus vulgaris L., is an important and valuablevegetable crop. Thus, a continuing goal of plant breeders is to developstable, high yielding garden bean cultivars that are agronomicallysound. The reasons for this goal are obviously to maximize the amount ofyield produced on the land. To accomplish this goal, the garden beanbreeder must select and develop garden bean plants that have the traitsthat result in superior cultivars.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel garden beancultivar, designated ‘210104’. This invention thus relates to the seedsof garden bean cultivar ‘210104’, to the plants of garden bean cultivar‘210104’ and to methods for producing a garden bean plant produced bycrossing the garden bean ‘210104’ with itself or another garden beanline, and to methods for producing a garden bean plant containing in itsgenetic material one or more transgenes and to the transgenic gardenbean plants produced by that method. This invention also relates tomethods for producing other garden bean cultivars derived from gardenbean cultivar ‘210104’ and to the garden bean cultivar derived by theuse of those methods. This invention further relates to hybrid gardenbean seeds and plants produced by crossing the line ‘210104’ withanother garden bean line.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of garden bean cultivar ‘210104’. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing gardenbean plant, and of regenerating plants having substantially the samegenotype as the foregoing garden bean plant. Preferably, the regenerablecells in such tissue cultures will be embryos, protoplasts, seeds,callus, pollen, leaves, anthers, roots, and meristematic cells. Stillfurther, the present invention provides garden bean plants regeneratedfrom the tissue cultures of the invention.

Another objective of the invention is to provide methods for producingother garden bean plants derived from garden bean cultivar ‘210104’.Garden bean cultivars derived by the use of those methods are also partof the invention.

The invention also relates to methods for producing a garden bean plantcontaining in its genetic material one or more transgenes and to thetransgenic garden bean plant produced by that method.

In another aspect, the present invention provides for single geneconverted plants of ‘210104’. The single transferred gene may preferablybe a dominant or recessive allele. Preferably, the single transferredgene will confer such trait as male sterility, herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, enhanced nutritional quality and industrial usage. Thesingle gene may be a naturally occurring bean gene or a transgeneintroduced through genetic engineering techniques.

The invention further provides methods for developing bean plant in abean plant breeding program using plant breeding technique includingrecurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection and transformation. Seeds, bean plant, and parties thereofproduced by such breeding methods are also part of the invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of a line are recovered in addition to thesingle gene transferred into the line via the backcrossing technique orvia genetic engineering.

Maturity Date. Plants are considered mature when the pods have reachedtheir maximum allowable seed size and sieve size for the specific useintended. This can vary for each end user, e.g., processing at differentstages of maturity would be required for different types of consumerbeans such as “whole pack,” “cut” or “french style”. The number of daysare calculated from a relative planting date which depends on daylength, heat units and environmental other factors.

Early maturity. Means less than 53 days is considered early, 54-59 dayswould be considered average maturity and 60 or more days would be latematurity.

Sieve Size (sv). Sieve size 1 means pods that fall through a sievegrader which culls out pod diameters of 4.76 cm through 5.76 cm. Sievesize 2 means pods that fall through a sieve grader which culls out poddiameters of 5.76 cm through 7.34 cm. Sieve size 3 means pods that fallthrough a sieve grader which culls out pod diameters of 7.34 cm through8.34 cm. Sieve size 4 means pods that fall through a sieve grader whichculls out pod diameters of 8.34 cm through 9.53 cm. Sieve size 5 meanspods that fall through a sieve grader which culls out pod diameters of9.53 cm through 10.72 cm. Sieve size 6 means pods that fall through asieve grader that will cull out pod diameters of 10.72 cm or larger.

Bean Yield (Tons/Acre). The yield in tons/acre is the actual yield ofthe bean pods at harvest.

Plant Height. Plant height is taken from the top of soil to top node ofthe plant and is measured in centimeters.

Field holding ability. A bean plant that has field holding ability meansa plant having pods that remain smooth and retain its color even afterthe seed is almost fully developed.

Machine harvestable bush. A machine harvestable bush means a bean plantthat stands with pods off the ground. The pods can be removed from theplant without leaves and other plant parts.

Plant adaptability. A plant having a good plant adaptability means aplant that will perform well in different growing conditions andseasons.

Emergence. Emergence is the rate that the seed germinates and sproutsout of the ground.

Plant architecture. Plant architecture is the shape of the overall plantwhich can be tall-narrow, short-wide, medium height, medium width.

Plant habit. Plant habit can be an upright plant or can be sprawling onthe ground.

Medium Pod set height. The pod set height is the location of the podswithin the plant. The pods can be high (near the top), low (near thebottom), or medium (in the middle) of the plant.

DETAILED DESCRIPTION OF THE INVENTION

Garden bean cultivar ‘210104’ has superior characteristics and wasdeveloped from the cross W6742*W6750, which was made 1995 in thegreenhouse at Harris Moran Research Station in Sun Prairie, Wis. The F₁hybrids were grown in a greenhouse during the Spring of 1996, also inSun Prairie (plot W6976). F₂ selection, plot number TA8202, was made atSan Juan Bautista, Calif. in the summer of 1996. The F₃ selections weremade in the winter of 1997 at Homestead, Fla. F₄ plants were selected ina field plot in California in summer 1997, F₅ selections were made inthe summer of 1998 in Sun Prairie, Wis. F₆ generation was selected inthe winter of 1999 in Homestead, Fla. F₇ plants were selected in agreenhouses in Sun Prairie during spring 1999 and the F₈ plants wereselected and then named ‘210104’ during the summer of 1999 in San JuanBautista, Calif.

The cultivar ‘210104’ is most similar to the variety ‘Hialeah’. ‘210104’has a greener pod than ‘Hialeah’. The pods of ‘210104’ remain smoothwhen they are past market maturity, while the pods of ‘Hialeah’ willbecome rough and bumpy. This field holding ability is a very importantcharacteristic of the pods of ‘210104’. The pods of ‘210104’ areoval-round in shape while the pods of ‘Hialeah’ are oval. The yield of‘210104’ will normally exceed the yield of ‘Hialeah’

‘210104’ is a green early maturing snap bean with medium green,straight, smooth fresh market pods, with a medium pod set height, whichare on a machine harvestable bush. ‘210104’ is resistant to Bean CommonMosaic Virus. In trials in the Eastern United States ‘210104’ has shownexcellent fresh pod yielding ability and plant adaptability. ‘210104’has consistently shown higher yields than commercial checks in theEastern United States. ‘210104’ has shown the ability of the pods toremain smooth and hold their color for several days after marketmaturity. This field holding ability, along with the early maturity, isa big advantage for fresh market growers.

Some of the criteria used to select in various generations include: podappearance and length, bean yield, pod set height, emergence, maturity,plant architecture, habit and height, seed yield and quality, anddisease resistance.

Bean common mosaic virus resistance is a desired trait for a beanvariety. The disease occurs worldwide causing low quality of the harvestproduct and losses from 80 to 100% by reduction of yield. It is mostlytransmitted by aphids during the growing season, but can also be spreadby pollen or mechanically. Leaves develop mosaic patterns in whichirregular light and dark green patch are intermixed. Malformation andyellow dots may also be produced, often causing growth reduction. Theplant may be dwarfed and pod and seed yield reduced. Severe necrosis mayoccur and the plant may die if infected while young. Systemic necrosis,in which the roots and shoots become blackened, appears in cultivarhaving dominant resistance gene/(hypersensitive resistance mechanism).The systemic necrosis may spread to higher leaves without killing themor may be concentrated in the vascular parts of the stem, eventuallyleading to the death of all or part of the plant. When infection occurslate in plant development, parts of the plant may die and many pods mayshow brown discoloration in the pod wall and pod suture as a result ofvascular necrosis.

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in ‘210104’.

Garden bean cultivar ‘210104’ has the following morphologic and othercharacteristics (based primarily on data collected at Sun Prairie, Wis.Research Station).

VARIETY DESCRIPTION INFORMATION

Market Maturity

-   Days to edible pods: 52 days.-   Number of days earlier than ‘Hialeah’: 1 day    Plant-   Habit: Determinate    Plant:-   Height: 50 cm-   Taller than ‘Hialeah” by 2 cm-   Spread: 48 cm-   Narrower than ‘Hialeah’ by 2 cm-   Pod position: Scattered-   Bush Form: Wide bush form    Leaves-   Surface: Dull-   Size: Medium-   Color: Medium Green    Anthocyanin Pigment-   Flowers: Absent-   Stems: Absent-   Pods: Absent-   Seeds: Absent-   Leaves: Absent-   Petioles: Absent-   Peduncles: Absent-   Nodes: Absent    Flower Color-   Color of standard: White-   Color of wings: White-   Color of keel: White    Pods (Edible Maturity)-   Exterior color: Medium-Green-   Dry pod color: Buckskin-   Cross Section Pod shape: Oval round-   Creaseback: Present-   Pubescence: Considerable-   Constriction: None-   Spur length: 10 mm-   Fiber: Considerable-   Number seeds/pod: 6-   Suture string: Absent-   Seed development: Medium-   Machine harvest: Adapted-   Distribution of Sieve Size at Optimum Maturity:-   25% 7.34-8.34 mm—Sieve 3-   65% 8.34-9.53 mm—Sieve 4-   10% 9.53-10.72—Sieve 5    Seed Color-   Seed coat luster: Shiny-   Seed coat: Monochrome-   Primary color: white-   Seedcoat Pattern: Solid-   Hilar ring: Absent    Seed Shape and Size-   Hilum view: Oval-   Cross section: Round-   Side view: Oval to Oblong-   Seed size: 26 gm/100 seeds-   2/100 gm seeds lighter than ‘Hialeah’    Disease Resistance-   Bean Common Mosaic Virus (BCMV) Resistant

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a garden plantby crossing a first parent bean plant with a second parent bean plantwherein either the first or second parent bean plant is an bean plant ofthe line ‘210104’. Further, both first and second parent bean plants cancome from the cultivar ‘210104’. Still further, this invention also isdirected to methods for producing a cultivar ‘210104’-derived bean plantby crossing cultivar ‘210104’ with a second bean plant and growing theprogeny seed, and repeating the crossing and growing steps with thecultivar ‘210104’-derived plant from 0 to 7 times. Thus, any suchmethods using the cultivar ‘210104’ are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using cultivar ‘210104’ as a parent are within thescope of this invention, including plants derived from cultivar‘210104’. Advantageously, the cultivar is used in crosses with other,different, cultivars to produce first generation (F₁) bean seeds andplants with superior characteristics.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which garden bean plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, pods, stems, roots, anthers, and the like.

As is well known in the art, tissue culture of garden bean can be usedfor the in vitro regeneration of a garden bean plant. Tissue culture ofvarious tissues of garden beans and regeneration of plants therefrom iswell known and widely published. For example, reference may be had toMcClean, P.; Grafton, K. F. (1989): “Regeneration of dry bean (Phaseolusvulgaris) via organogenesis.” Plant Sci. 60,117-122. Mergeai, G.;Baudoin, J. P. (1990): “Development of an in vitro culture method forheart-shaped embryo in Phaseolus vulgaris.” B.I.C. Invit. Papers 33,115-116. Vanderwesthuizen, A. J.; Groenewald, E. G. (1990): “RootFormation and Attempts to Establish Morphogenesis in Callus Tissues ofBeans (Phaseolus-Vulgaris L.).” S. Afr. J. Bot. 56(2, April), 271-273.Benedicic, D., et al. (1990): “The regeneration of Phaseolus vulgaris L.plants from meristem culture.” Abst. 5th I.A.P.T.C. Cong. 1, 91(#A3-33). Genga, A.; Allavena, A. (1990): “Factors affectingmorphogenesis from immature cotyledons of Phaseolus coccineus L.” Abst.5th I.A.P.T.C. Cong. 1, 101 (#A3-75). Vaquero, F., et al. (1990): “Plantregeneration and preliminary studies on transformation of Phaseoluscoccineus.” Abst. 5th I.A.P.T.C. Cong. 1, 106 (#A3-93). Franklin, C. I.,et al. (1991): “Plant Regeneration from Seedling Explants of Green Bean(Phaseolus-Vulgaris L.) via Organogenesis.” Plant Cell Tissue Org. Cult.24(3, March), 199-206. Malik, K. A.; Saxena, P. K. (1991): “Regenerationin Phaseolus-Vulgaris L.—Promotive Role of N6-Benzylaminopurine inCultures from Juvenile Leaves.” Planta 184(1), 148-150. Genga, A.;Allavena, A. (1991): “Factors affecting morphogenesis from immaturecotyledones of Phaseolus coccineus L.” Plant Cell Tissue Org. Cult. 27,189-196. Malik, K. A.; Saxena, P. K. (1992): “Regeneration in Phaseolusvulgaris L.L.—High-Frequency Induction of Direct Shoot Formation inIntact Seedlings by N-6-Benzylaminopurine and Thidiazuron.” 186 (3,February), 384-389. Malik, K. A.; Saxena, P. K. (1992): “SomaticEmbryogenesis and Shoot Regeneration from Intact Seedlings of Phaseolusacutifolius A., P. aureus (L.) Wilczek, P. coccineus L., and P. wrightiiL.” PI. Cell. Rep. 11 (3, April), 163-168. Chavez, J., et al. (1992):“Development of an in vitro culture method for heart shaped embryo inPhaseolus polyanthus.” B.I.C. Invit. Papers 35, 215-216. Munoz-Florez,L. C., et al. (1992): “Finding out an efficient technique for inducingcallus from Phaseolus microspores.” B.I.C. Invit. Papers 35, 217-218.Vaquero, F., et al. (1993): “A Method for Long-Term Micropropagation ofPhaseolus coccineus L.” L. PI. Cell. Rep. 12 (7-8, May), 395-398. Lewis,M. E.; Bliss, F. A. (1994): “Tumor Formation and beta-GlucuronidaseExpression in Phaseolus vulgaris L. Inoculated with AgrobacteriumTumefaciens.” Journal of the American Society for Horticultural Science119 (2, March), 361-366. Song, J. Y., et al. (1995): “Effect of auxin onexpression of the isopentenyl transferase gene (ipt) in transformed bean(Phaseolus vulgaris L.L.) single-cell clones induced by Agrobacteriumtumefaciens C58.” J. Plant Physiol. 146 (1-2, May), 148-154. It is clearfrom the literature that the state of the art is such that these methodsof obtaining plants are, and were, “conventional” in the sense that theyare routinely used and have a very high rate of success. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce bean plants having the physiological andmorphological characteristics of variety ‘210104’.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed bean plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the bean plant(s).

Expression Vectors for Bean Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983), Aragao F. J. L., et al., Molecular Breeding 4:6491-499 (1998). Another commonly used selectable marker gene is thehygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988), Saker M. M., etal, Biologia Plantarum 40:4 507-514 (1998), Russel, D. R., et al, PlantCell Report 12:3 165-169 (1993).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include beta-glucuronidase (GUS), alpha-galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984), Grossi M. F., et al., Plant Science 103 :2189-198 (1994), Lewis M. E., Journal of the American Society forHorticultural Science 119:2 361-366 (1994), Zhang et al., Journal of theAmerican Society for Horticultural Science 122:3 300-305 (1997).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inbean. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in bean. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inbean or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in bean.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985), Aragao et al., Genetics andMolecular Biology 22:3, 445-449 (1999) and the promoters from such genesas rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730(1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300(1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin bean. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in bean. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is bean. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence ofδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo α-1, 4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to a Herbicide, for Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. See also Russel, D. R., et al, Plant Cell Report12:3 165-169 (1993). The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Delayed and attenuated symptoms to Bean Golden Mosaic Geminivirus(BGMV), for example by transforming a plant with antisense genes fromthe Brazilian BGMV. See Arago et al., Molecular Breeding. 1998, 4:6,491-499.

B. Increased the bean content in Methionine by introducing a transgenecoding for a Methionine rich storage albumin (2S-albumin) from theBrazil nut as decribed in Arago et al., Genetics and Molecular Biology.1999, 22: 3, 445-449.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). McClean, P., et al. Plant CellTissue Org. Cult. 24(2, February), 131-138 (1991), Lewis et al., Journalof the American Society for Horticultural Science 119:2, 361-366 (1994),Zhang, Z., et al. J. Amer. Soc. Hort. Sci. 122(3): 300-305 (1997). A.tumefaciens and A. rhizogenes are plant pathogenic soil bacteria whichgenetically transform plant cells. The Ti and Ri plasmids of A.tumefaciens and A. rhizogenes, respectively, carry genes responsible forgenetic transformation of the plant. See, for example, Kado, C. I.,Crit. Rev. Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by Gruber et al., supra, Miki et al., supra, and Moloney etal., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No. 5,591,616issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal or vegetable crop species andgymnosperms have generally been recalcitrant to this mode of genetransfer, even though some success has recently been achieved in riceand corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat.No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 ìm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al. Pl. Cell. Rep. 12(3, January), 165-169 (1993), Aragao, F. J. L.,et al. Plant Mol. Biol. 20(2, October), 357-359 (1992), Aragao Theor.Appl. Genet. 93: 142-150 (1996), Kim, J.; Minamikawa, T. Plant Science117: 131-138 (1996), Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992)

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet.199:161(1985) and Draperet al., Plant Cell Physiol. 23:451(1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994).

Following transformation of bean target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic bean line. Alternatively, a genetic trait which hasbeen engineered into a particular bean cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

When the term garden bean plant, cultivar or bean line are used in thecontext of the present invention, this also includes any single geneconversions of that line. The term single gene converted plant as usedherein refers to those garden bean plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a cultivarare recovered in addition to the single gene transferred into the linevia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into theline. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental bean plants forthat line. The parental bean plant which contributes the gene for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental bean plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a gardenbean plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,herbicide resistance (such as bar or pat genes), resistance forbacterial, fungal, or viral disease such as gene I used for BCMVresistance), insect resistance, enhanced nutritional quality (such as 2salbumine gene), industrial usage, agronomic qualities such as the“persistent green gene”, yield stability and yield enhancement. Thesegenes are generally inherited through the nucleus. Some other singlegene traits are described in U.S. Pat. Nos. 5,777,196; 5,948,957 and5,969,212, the disclosures of which are specifically hereby incorporatedby reference.

TABLES

In Table 1 and 2 that follows, the traits and characteristics of gardenbean line ‘210104’ are given along with data on a commercial garden beanline used as a check.

The first column lists the variety tested.

The second column shows the location.

Columns 3 shows the plant height in cm.

Columns 4 shows the pod length in cm.

Column 5 shows the pod color from 1 light to 5 dark.

Column 6 shows days to maturity.

Column 7 shows the yield in pounds per 5 feet plot.

TABLE 1 Overall Comparisons Garden Bean named ‘210104’ vs ChecksLocation: 2001 Plt Pod Pod Variety Location Height Length Color MaturityYield Hialeah Georgia 53 16 1.5 52 5.5 Hialeah Georgia 49 16 1.5 51 6.31‘210104’ Georgia 49 16 2.5 51 4.13 ‘210104’ Georgia 55 16 2 52 5 HialeahHomestead, FL 40 15 1.5 52 2.3 ‘210104’ Homestead, FL 40 15 2 52 2.5Hialeah Tennessee 50 15 1.5 62 3.44 ‘210104’ Tennessee 55 15 2 62 4.19

TABLE 2 Overall Comparisons Garden Bean named ‘210104’ vs ChecksLocation: 2000 Plt Pod Pod Matu- Variety Location Height Length Colorrity Yield Hialeah Crossville 50 16 1.5 50 3.2 Hialeah Crossville 53 151.5 50 2.8 ‘210104’ Crossville 50 15 2 50 4.4 ‘210104’ Crossville 50 152.5 50 4.1 Hialeah Delmarva 56 14.5 1 57 3 ‘210104’ Delmarva 54 14 1 553.8 Hialeah Homestead 1, FL 50 16 1.5 52 2.13 ‘210104’ Homestead 1, FL58 15 2.5 52 2.5 Hialeah Homestead 2, FL 50 16 1.5 52 2.19 ‘210104’Homestead 2, FL 50 15 2.5 53 2.19 Hialeah Homestead 3, FL 38 16 1.5 571.9 ‘210104’ Homestead 3, FL 40 15 2.5 60 2.5 Hialeah Homestead 4, FL 4515 1.5 54 2.2 ‘210104’ Homestead 4, FL 44 15 2 54 2.8 Hialeah New York55 15 1 58 3.8 ‘210104’ New York 53 16 2 60 4.6 Hialeah Swan Quarter 4816 1 58 2.69 ‘210104’ Swan Quarter 46 14.5 1.5 59 4.88

DEPOSIT INFORMATION

A deposit of the Harris Moran Seed Company proprietary garden beancultivar named ‘210104’ disclosed above and recited in the appendedclaims has been made with National Collections of Industrial Food andMarine Bacteria (NCIMB), 23 St. Machar Drive, Aberdeen, Scotland, AB243RY, United Kingdom. The date of deposit was Nov. 8, 2004. The depositof 2,500 seeds was taken from the same deposit maintained by HarrisMoran Seed Company since prior to the filing date of this application.All restrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801-1.809. TheNCIMB accession No. for garden bean cultivar named ‘210104’ is NCIMB41260. The deposit will be maintained in the depository for a period of30 years, or 5 years after the last request, or for the effective lifeof the patent, whichever is longer, and will be replaced as necessaryduring that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somaclonal variants, variant individuals selected from large populationsof the plants of the instant line and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

1. A Phaseolus vulgaris L. garden bean seed designated ‘210104’, whereina sample of said seed has been deposited under NCIMB No.
 41260. 2. Aplant, or its parts, produced by growing the seed of claim
 1. 3. Pollenof the plant of claim
 2. 4. An ovule of the plant of claim
 2. 5. APhaseolus vulgaris L. garden bean plant, or its parts, having all of thephysiological and morphological characteristics of the garden bean plantof claim
 2. 6. A tissue culture of regenerable cells of a bean plant ofvariety ‘210104’, wherein the tissue regenerates plants capable ofexpressing all the morphological and physiological characteristics ofPhaseolus vulgaris L. bean line ‘210104’, representative seeds havingbeen deposited under NCIMB No.
 41260. 7. The tissue culture of claim 6,selected from the group consisting of protoplast and calli, wherein theregenerable cells are derived from embryo, meristematic cells, leaves,pollen, embryo, root, root tips, stems, anther, flowers, seeds or pods.8. A Phaseolus vulgaris L. garden bean plant regenerated from the tissueculture of claim 6, wherein the regenerated plant has all themorphological and physiological characteristics of Phaseolus vulgaris L.bean plant ‘210104’, representative seeds having been deposited underNCIMB No.
 41260. 9. A method for producing a garden bean seed comprisingcrossing a first parent garden bean plant with a second parent gardenbean plant and harvesting the resultant hybrid garden bean seed, whereinsaid first or second parent garden bean plant is the Phaseolus vulgarisL. garden bean plant of claim
 2. 10. A method of producing an herbicideresistant bean plant comprising transforming the bean plant of claim 2with a transgene that confers herbicide resistance.
 11. An herbicideresistant bean plant produced by the method of claim
 10. 12. The beanplant of claim 10, wherein the transgene confers resistance to anherbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 13. A method of producing an insect resistant bean plantcomprising transforming the bean plant of claim 2 with a transgene thatconfers insect resistance.
 14. An insect resistant bean plant producedby the method of claim
 13. 15. The bean plant of claim 14, wherein thetransgene encodes a Bacillus thuringiensis protein.
 16. A method ofproducing a disease resistant bean plant comprising transforming thebean plant of claim 2 with a transgene that confers resistance tobacterial, fungal or viral disease.
 17. A disease resistant bean plantproduced by the method of claim
 16. 18. A method of introducing adesired trait into bean cultivar ‘210104’ comprising: (a) crossing thebean cultivar ‘210104’ plants grown from the bean cultivar ‘210104’seed, representative seed of which has been deposited under NCIMB No.41260 with plants of another bean cultivar that comprise a desired traitto produce F1 progeny plants, wherein the desired trait is selected fromthe group consisting of herbicide resistance, insect resistance,resistance to bacterial disease, resistance to fungal disease orresistance to viral disease; (b) selecting F1 progeny plants that havethe desired trait to produce selected F1 progeny plants; (c) crossingthe selected F1 progeny plants with the bean cultivar ‘210104’ plants toproduce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait and physiological and morphologicalcharacteristics of bean cultivar ‘210104’ listed in the VarietyDescription Information to produce selected backcross progeny plants;and (e) repeating steps (c) and (d) three or more times in succession toproduce selected second or higher backcross progeny plants that comprisethe desired trait and the physiological and morphologicalcharacteristics of bean cultivar ‘210104’ listed in the VarietyDescription Information as determined at a 5% significance level whengrown in the same environmental conditions.
 19. A bean plant produced bythe method of claim 18, wherein the plant has the desired trait and thephysiological and morphological characteristics of bean cultlvar‘210104’ listed in the Variety Description Information as determined ata 5% significance level when grown in the same environmental conditions.